Journal of Food Hygiene and Safety (한국식품위생안전성학회지)

The Korean Society of Food Hygiene and Safety (한국식품위생안전성학회)
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Detection of Salmonella spp. in Seafood via Desalinized DNA Extraction Method and Pre-culture

전배양과 탈염과정을 포함하는 DNA 추출법을 이용한 분자생물학적 방법으로 수산물 중 오염된 Salmonella spp.의 검출

  • Ye-Jun Song (Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety) ;
  • Kyung-Jin Cho (Department of Food Science and Technology, Pukyong National University) ;
  • Eun-Ik Son (Food Standard Division, Food Standard Planning Office, Ministry of Food and Drug Safety) ;
  • Du-Min Jo (Department of Food Science and Technology, Pukyong National University) ;
  • Young-Mog Kim (Division of Food Science, Pukyoung National Universitry) ;
  • Seul-Ki Park (Smart Food Manufacturing Project Group, Korea Food Research Institute)
  • 송예준 (식품의약품안전처 식품의약품안전평가원 식품위해평가부 미생물과) ;
  • 조경진 (부경대학교 식품공학과) ;
  • 손은익 (식품의약품안전처 식품의약품안전처본부 식품기준기획관 식품기준과 ) ;
  • 조두민 (부경대학교 식품공학과) ;
  • 김영목 (부경대학교 식품과학부) ;
  • 박슬기 (한국식품연구원 스마트제조사업단)
  • Received : 2023.04.10
  • Accepted : 2023.05.02
  • Published : 2023.06.30
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Abstract

Salmonella spp. are prevalent foodborne pathogens that are infective at relatively low concentrations, thus posing a serious health threat, especially to young children and the elderly. In several countries, the management and regulation of Salmonella spp. in food, including seafood, adhere to a negative detection standard. The risk of infection is particularly high when seafood is consumed raw, which underscores the importance of timely detection of pathogenic microorganisms, such as Salmonella. Accordingly, this study aimed to develop a combined pre-treatment and detection method that includes pre-culture and DNA extraction in order to detect five species of Salmonella at concentrations below 10 CFU/mL in seafood. The effectiveness of the proposed method was assessed in terms of the composition of the enrichment (pre-culture) medium, minimum incubation time, and minimum cell concentration for pathogen detection. Furthermore, a practical DNA extraction method capable of effectively handling high salt conditions was tested and found to be successful. Through polymerase chain reaction, Salmonella spp. Were detected and positively identified in shellfish samples at cell concentrations below 10 CFU/g. Thus, the proposed method, combining sample pre-treatment and cell culture with DNA extraction, was shown to be an effective strategy for detecting low cellular concentrations of harmful bacteria. The proposed methodology is suitable as an economical and practical in situ pre-treatment for effective detection of Salmonella spp. in seafood.

본 연구에서는 수산물 시료 중 Salmonella spp. 검출을 위해 단시간의 전배양(2시간 이내)과 탈염과정을 포함한 DNA 추출법을 사용하여 분자생물학적 검출을 위한 수산물 전처리 방법에 대해 연구하였다. 배양 시간에 따른 증균 효율을 탐색하기 위해 100, 101 및 102 CFU/mL농도의 Salmonella spp. 5종을 NB 0.5에 접종하여 증균 전, 1시간 및 2시간 동안의 증균 효율을 비교하였다. 그 결과, 2시간 동안 모든 농도에서 약 1 log CFU/mL가 증균되어 초기 농도와 유의적인 차이가 나타났다. 또한 지역별 패류시료에 S. Typhimurium을 인위적으로 감염시킨 뒤 DNA를 추출하여 염농도를 측정한 결과, 모든 시료의 염농도가 0%로 DNA 추출과 동시에 탈염이 이루어진 것을 확인하였다. 이후 추출한 DNA를 사용하여 PCR을 수행한 결과 모든 시료에서 S. Typhimurium의 특이적 양성밴드가 확인되었다. 다음으로 수산물 시료 중 Salmonella spp. 검출을 위한 증균 과정과 탈염을 포함한 DNA 추출방법의 검증을 위해 멸균 홍합시료 및 비멸균 홍합시료에 Salmonella spp. 5종을 인공적으로 약 100, 101, 102 CFU/g의 농도로 오염시켜 전배양과 DNA를 추출하여 PCR로 특이적 증폭 밴드의 여부를 확인한 결과, 모든 농도의 Salmonella spp. 5종에서 특이적 밴드가 확인되었다. 결과적으로 본 연구에서 제시한 전배양 및 DNA 추출방법을 포함한 전처리 방법과 PCR을 사용하여 수산물 시료에서 10 CFU/g 미만의 Salmonella spp.를 검출하였으며, 시간과 비용면에서 효율적이며 과정이 복잡하기 않기 때문에 수산물의 처리 현장에 활용될 수 있을 것으로 기대된다.

Keywords

Acknowledgement

이 논문은 2023년도 해양수산부 재원으로 해양수산과학기술진흥원의 지원을 받아 수행된 연구임(20210695 수산물 신선유통 스마트 기술개발). 이 논문은 2020년도 해양수산부 재원으로 해양수산과학기술진흥원의 지원을 받아 수행된 연구임(20200377 수산 환경 및 수산물 중 미생물학적 위해요소 현장형 진단시스템 개발).

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